Proteins with a high immunoreactivity and a method for the production thereof

ABSTRACT

The invention relates to (glyco-) proteins, in particular monoclonal antibodies, which have an immunoreactivity of &gt;81%, preferably &gt;90%. The inventive monoclonal antibodies are produced using a fluidized bed reactor in conjunction with a conventional protein-chemical purification method or preferably with a purification method involving less column chromatography. The monoclonal antibodies thus produced are suitable, in gamma-irradiated form, e.g. Tc-99m labelled, for the in vivo diagnosis of inflammatory diseases and bone marrow metastases. In alpha - or beta-irradiated form, e.g. astatine or Re-188 or Y-90 labelled form, the inventive monoclonal antibodies can be used, for example, in the treatment of leukemia.

[0001] The invention relates to preparations of immunoreactive proteins and in particular in purified form and having a high percentage of immunoreactive molecules relative to the total number of molecules. These proteins can be obtained through fermentation in a fluidized reactor of a host cell, which is capable of expressing the immunoreactive protein and production of the protein from the host cell respectively from the culture medium used for culturing the cells. The protein preparations according to the invention are outstandingly suitable for producing diagnostic and therapeutic compositions.

[0002] Diagnostic and therapeutic proteins can be expressed in prokaryotic cells, such as for example E. coli (Houston et al., U.S. Pat. No. 5,132,405) as well as in eukaryotic cell systems, (for example Pichia pastoris, baby hamster kidney (BHK) cells, Chinese hamster Ovary (CHO-)cells, hybridomas, transgenic animals and plants (Meade et. al., U.S. Pat. No. 4,873,316) and can be purified by protein-chemical methods.

[0003] With prokaryotic cell systems, a cost efficient production of small carbohydrate-free proteins can be realized due to the simple culture media and the fast growth of the microorganisms in relatively simple fermentation systems.

[0004] However, for the production of complex, carbohydrate-carrying (glyco-) proteins, the afore-described eucaryotic cells systems have to be used which require complex fermentation systems and require the use of expensive culture media. The compositions of the glyco- protein preparation that are to be purified are being decisively influenced by the type of culture medium and the fermentation system used. In particular, differences of the glycosylation of the protein, which influences the half-life in the plasma as well as certain biological effector functions of the purified (glyco-) protein preparation are described in the resepctive technical literature. (Stahl et al., PNAS, 73 (1976), 4045-4049).

[0005] In addition to the afore-described influences on the carbohydrate composition, an essential factor which determines the quality of the product is the percentage share of functionally active (glyco-) proteins in the purified preparation, which can be also influenced by the type of cell media used, the conditions of fermentation and the purification methods applied.

[0006] As a preferred method used for determining the portion of functionally active radio-labeled molecules (for example MAk with physiological intact V-region) is the quantitative binding assay first described by Lindmo et al. (Immunological Methods 72 (1984) 77) in which an excess of antigen was used. This methods permits a quantitative determination of the immunoreactive and radioactive labeled MAk (monoclonal antibody) portion having a bonding capacity, after protein chemical purification. Other binding assays, such as the immunoreactivity test of Seitz et al. (European Journal of Nuclear Medicine 26 (1999), 1265) where immunoreactivities of up to 100% are described, permit only a relative determination of the immunoreactive portion of radio-labeled MAk in comparison to the unlabelled standard and are thus not suitable for determination of the absolute immunoreactive fraction. Similarly, the method described by Jagoda et al. (Journal of Immunological Methods 173 (1994), 191-201) directed to an affinity-chromatographic method for determining immunoreactivity has only limited applicability for a quantitative determination of the immunoreactive portion due to the relatively high portion of unspecific linkage with the affinity column (10-20%).

[0007] Likewise, the Lindmo-test utilized by most authors, and despite the authors claim that the method has general applicability, it is only suitable for the evaluation of radio-labeled MAk where binding can be realized to a distinct plateau by utilizing increasing amounts of antigen. Oftentimes, a distinct plateau is not reached with MAks of low avidity or with a lack of cells that express high antigen densities per cell (Mattes, M. J., Int. J. Cancer: 61 (1995), 286-288) Even in investigations, where a definitive plateau can be reached, plotting the data from the saturation curves according to the Lindmo et al. method, produces two straight crossed lines, where the intersection with the Y-axis can lead to differing immunoreactivities. (Mattes, M. J., Int. J. Cancer: 61 (1995), 286-288, page 287, FIGS. 1B and E). Thus, when using the Lindmo method the maximal specifically linked radioactive portion and the value extrapolated to the infinite antigen excess must be given. When the difference between these two values is >10%, then the extrapolated immunoreactivity value cannot be regarded as reliable (Mattes M. J., Int. J. Cancer: 61 (19915), 286-288, page 288).

[0008] With regard to the afore-described reservations concerning that method, when using the Lindmo test, the percent portion of the functionally active MAk molecule prepared with conventional fermenting methods, such as for example stirred fermentors and hollow fiber modules and purified with classical protein chemical methods which also comprise, besides protein A-columns, io exchanger columns and in many cases also gel-chromatographic columns, is in the range of 40-80 % (Mattes, M. J., Int. J. Cancer: 61 (1995), 286-288; Jagoda et al., Journal of Immunological Methods 173 (1994), 191-201; Morales-Morales et al., Nuclear Medicine & Biology, Vol. 26 (1999), 275-279; Boven et al., Blood, 67, N9 2 (1986), 429-435). Here, the radio-labeling method and the radioisotope used likewise have an important role.

[0009] This means that 20-60% of the MAk molecules of these prepared preparations do not carry out the desired diagnostic or therapeutic function due to their production method.

[0010] One exception known in the literature is the granulascint described by Schubinger et al., (Eur. J. Nucl. Med. 15: (1989), 605-608) where an immunoreactivity of 93 respectively 40% is described, depending on the respective labeling methods. However, this MAk was produced in the form of ascites (personal communication). This in vivo method fulfills neither economic nor modern regulatory requirements and therefor has to be regarded as an exception. Furthermore, it is noted, that the portion of the really specifically binding MAk molecules is smaller than the value determined in the Lindmo test. This is the result of the unspecific binding of radio-labeled MAk to e. g. the granulocytes used in an antigen excess even with the addition of a 10 000 times molar excess of unlabelled MAk (Schubinger et al., Euro J. Nucl. Med., 15 (1989), 605-608). Accordingly, after subtraction of unspecific binding the immunoreactivity value is at <90%.

[0011] In the case of radio-labeled diagnostic monoclonal antibodies, a reduced immunoreactivity of the MAk is a definite disadvantage, since radio-labeled molecules that are not binding to the specific target structure circulate in the blood, contribute to the unspecific background are partially absorbed by the liver and make the interpretation of nuclear-medical diagnosis more difficult. In the case of radio-labeled therapeutic antibodies, the portion of the MAk molecules not bound to the target tissue contributes to the unspecific and undesired irradiation of non-target tissue.

[0012] It is thus of great importance in particular for producing radio-labeled monoclonal antibodies to provide economically efficient in vitro methods that pass regulatory acceptance and with which production of MAk preparations that have an as high as possible immunoreactive portion can be realized.

[0013] At this time, highly immunoreactive portions can only be realized by means of complex immunoaffinity-chromatographic methods which are not suitable for subsequent commercialization by the pharma industry. Here, the functionally active molecules are separated from the functionally inactive molecules which are protein-chemically not distinguishable from the active molecules, through binding to antigen columns whereby they are also concentrated. Despite the relatively high cost in the production of the reagents necessary for the immunoaffinity chromatography and the high losses during purification, the portion of the functionally active MAk in such preparations utilized for research purposes is even <81%.

[0014] In order to be able to measure with certainty the immunoreactive portion of the non-labeled MAk as well as the labeled MAk, a “modified Lindmo test” was developed, which is described infra in detail in Example 3. In this modified Lindmo test the portion of unspecifically binding MAk-molecules (as for example found after radio labeling with a 10 000-fold excess of cold MAk, Schubinger et al. Euro J. Nucl. Med., 15 (1989), 605-608) does not influence the computed immunoreactivity value.

[0015] Within the scope of researching an optimization of the production methods for the monoclonal antibody, BW 250/183 (Eur J. Nucl. Med. 14 (1988), 523-528 and Int. J. Cancer 36 (1985), 75-84) it was surprisingly possible to develop a fermentation method which produces cell culture supernatants which contain MAk with an immunoreactivity as shown by the modified Lindmo Test to be regularly >95%.

[0016] From these cell culture supernatants, MAk preparations can be produced by using conventional purification methods (also corresponding to GMP), and which exhibit an immunoreactivity between 80 and 90%. The loss in immunoreactivity observed here of about 10% during the purification is due to the multiple steps of column chromatography.

[0017] Furthermore, a “column-reduced” purification method was developed, with which MAk preparations can be produced (also GMP corresponding) that shows an immunoreactivity in the modified Lindmo test, which does not significantly differ from that of the non-purified MAk molecules found in the cell culture supernatant (loss of about 1-2%).

[0018] These methods can be applied to a variety of MAks of differing specificities and protein chemical compositions as well as to other immunoreactive proteins, and they produce also in these cases comparably surprising results.

[0019] The invention relates to a preparation of immunoreactive proteins, wherein the per cent portion of the immunoreactive molecules, determined by a modified Lindmo test is >81%, preferably >90% and most preferred >95% relative to the entire number of molecules. The immunoreactive proteins can be obtained by production in cell culture, in particular in eucaryotic host cells. The immunoreactive proteins can be coupled to markers, in particular a radioactive marker, without any loss of reactivity.

[0020] Immunoreactive proteins according to the invention are proteins that have at least one antibody-binding domain. Examples for such proteins are antibodies, in particular monoclonal antibodies, chimerical antibodies, humanized antibodies, recombinant antibodies such as single chain antibodies or fragments thereof, e.g. proteolytic fragments, such as Fab-, Fab′-or F(ab)₂-fragments or recombinant antibody fragments, such as single chain Fv-fragments.

[0021] In addition to antibodies and antibody fragments, fusion proteins can likewise be utilized, which comprise at least one antibody binding domain and a further binding domain, for example an effector domain, such as an enzyme or a cytokine. Examples thereof are scFv-Cytokine, e. g. scFv-IL1, scFv-IL2, scFv-IL6, scFv-IL10, scFv-IL11, scFv-IL12, scFv-TNF, scFv-IFNγ, scFv-IFNβ, scFv-IFNα, or scFv-coagulation agents, such as for example scFv-tTF, scFv-(Deoxy) ribonucleases or fusion with enzymes, such as the fusion protein described in British J. Cancer 654 (1992), 234-238 which comprises the heavy chain VH-CH1 of the humanized MAk BW 431/26 fused via hingelinker to β-glucuronidase and the humanized VL-CL light chain of the same humanized MAk.

[0022] The protein preparations in accordance with the invention are obtainable through fermentation in a fluidized bed. In such a fluidized bed, the cultured host cells, for example hybridoma—or other eucaryotic cells are grown in high density on glass spheres and can be optimally provided with oxygen and nutrients. Dying cells detach from the glass spheres and are continuously removed together with the harvested medium from the fermentation system. Thus, the amounts of proteases and cell debris can be kept to a minimum in the fermentation system whereby the integrity of the produced protein is realized. One example of a suitable fluidized-fermentation reactor is bioreactor Pilot B 500 (Papaspyrou Biotechnologie GmbH).

[0023] Measurement of the immunoreactivity of the protein preparations according to the invention is carried out by means of the modified Lindmo test described in Example 3. When carrying out this test, it is essential that an excess amount of the antigen containing material is used in order to quantitatively bind the immunoreactive protein, e. g. the MAk. Furthermore, it is important; that the antigen containing material contains a sufficient amount of epitopes, so that unspecific adsorption effects caused by large surfaces can be disregarded. Thus, epitope densities of >10⁴ per fixed cell are preferred. The immunoreactivity is read at the point at which the control MAk (same isotype as the test MAk, but having non-relevant binding region) does not show a significant unspecific adsorption.

[0024] The following describes in addition to the fermentation method according to the invention and the “column-reduced” purification method, also the protein preparations according to the invention that result therefrom and their advantageous use as diagnostic and therapeutic tools.

[0025] The two methods in accordance with the invention I and II are comprised in each case of two sections, which can be further subdivided into several single steps. Production Method I Production Method II (general) (general) Culturing of eucaryotic cells and their Culturing of eucaryatic cells and their fermentation in a fluidized reactor fermentation in a fluidized reactor Proteinchemical purification with a Proteinchemical purification with a conventional purification system “column reduced” purifi- cation method

[0026] Production Method I (Specific):

[0027] Culturing of Eucaryotic Cells and Their Fermentation in a Fluidized Reactor:

[0028] 1. defrosting cells from the cell bank and culturing for example in T-flasks

[0029] 2. expanding cells for example in Spinner cultures

[0030] 3 fermentation in the fluidized reactor

[0031] Protein-Chemical Purification with Conventional Purification Methods

[0032] 1. harvesting the cell culture medium, separating cells e. g. by means of microfiltration, sterile filtration and storage, for example at −20° C.;

[0033] 2. concentrating, for example by means of ammonium sulfate precipitation and subsequent centrifugation;

[0034] 3. dissolution; deactivating of virus by means of detergent treatment and sterile filtration;

[0035] 4. submitting to affinity-chromatography with protein A or similar affinity system;

[0036] 5. concentrating for example by means of ammonium sulfate precipitation and subsequent centrifugation

[0037] 6. dissolution, rebuffering by means of gel-chromatography

[0038] 7. anion exchange chromatography

[0039] 8. concentrating for example by means of ammonium sulfate precipitation and subsequent centrifugation;

[0040] 9 dissolution, rebuffering by means of gel-chromatography;

[0041] 10. adjusting the final desired concentration of the batch;

[0042] 11. sterile filtration.

[0043] Production Method II (Specific)

[0044] Culturing Eucaryotic Cells and Their Fermentation in a Fluidized Reactor

[0045] 1. defrosting cells from the cell bank and cultivating cells, for example in T-flasks;

[0046] 2. expanding cells, for example in spinner-cultures;

[0047] 3. Fermentation in the fluidized reactor.

[0048] Protein-Chemical Purification with a “column reduced” Purification Method

[0049] 1. harvesting of the cell culture medium, cell separation, for example by means of micro filtration, sterile filtration and storage, for example at −20° C.;

[0050] 2. defrosting of the sterile and cell-free culture, harvest followed by microfiltration and ultrafiltration;

[0051] 3. dilution;

[0052] 4. treatment with detergent;

[0053] 5. submitting to affinity-chromatography with protein A or similar affinity system;

[0054] 6. dilution and sterile filtration;

[0055] 7. anion exchange-membrane adsorption;

[0056] 8. virus filtration by means of ultrafiltration;

[0057] 9. ultrafiltration and diafiltration;

[0058] 10. filtration and dilution;

[0059] 11. sterile filtration.

[0060] The above shown individual production method steps are each known to the skilled artisan (cell biologists/protein chemists) and need not be further described. Notwithstanding that, the method is extensively described herein in Example 2.

[0061] Production method I is novel in that the fermentation is carried out with the fluidized reactor in combination with a conventional purification method.

[0062] Surprisingly, the immune reactivity of the MAk in unpurified cell culture supernatants (T-flasks, fluidized reactor) is >90% in the modified Lindmo test. In accordance with conventional purification, immunoreactivities of >80% are realized.

[0063] The novelty of production method II is the fermentation by means of a fluidized reactor in combination with the “column-reduced” purification method, wherein only one column purification chromatography (affinity chromatography at protein A) is utilized. All other steps are either gentle filtration steps by means of membranes or simple dilution steps. The method according to the present invention also fulfills the requirements of the respective governing approval agency concerning the protein chemical purity and the virus depletion and the inactivation.

[0064] Surprisingly, the immunoreactivity of the (glyco-) protein, for example an MAk, at all tested levels (T-Flasks, fluidized reactor, purified protein) is >90% in the Lindmo test, most often even 95% as shown in exemplary manner in Example 1 for the MAk BW 250/183. Comparatively high immunoreactivity values for in vitro production methods disclosed in the prior art have not been achieved.

[0065] Similarly high immunoreactivity values are found with the method according to the present invention for other MAks, such as for example MAk BW 431/26 (selective for CD 66e) (Eur. J. Nucl. Med. 14 (1988), 523-528 and Int. J. Cancer 36 (1985), 75-84), Mak BW 278/105 (selective for a subpopulation of FVIII RAG) (J. Histochem. Cytochem. 34 (1986), 209-214), MAk BW 575/931 (selective for N-CAM) ( . . . ) and MAk YTH 24.5 (selective for CD 45) (J. Immunol. 134 (1985), 3056-3061 and Leucocyte Typing III: White Cell Differentiation Antigens (released McMichael et al.), Oxford University Press, Oxford, pp 788-803 and several MAks described in the German patent application 197 44 531.4 (selective for the VEGF/VEGF-receptor complex, which however bind neither to the VEGF nor to the VEGF-receptor by itself). These findings show that with the production method according to the present invention, fermentation of (glyco-) proteins is realized, which are immunoreactive to approximately 100% and which can be purified gently and rapidly, in the case of production method II without any traceable loss in immunoreactivity and in an economically efficient manner.

[0066] Furthermore, the production methods according to the present invention for the GMP-commensurate production of a multitude of (glyco-) proteins having no Fc-portions can be utilized, among others, for the fusion protein described by Bosslet et al. (British Journal of Cancer, 645 (1992), 234-238), wherein instead of the A-protein affinity chromatography, an alternative affinity chromatography procedure was utilized (anti-idiotype, lectin column, protein L, nickel acid etc.)

[0067] In addition to testing the immunoreactivity, a number of quality controls are conducted at the respective production levels such as for example tests for sterility, DNA content, protein content, specificity, pH value, protein composition, iso-electric point, pyrogenes, protein A—content in the end product), which permit testing the microbiological, immunological and proteinchemical properties of the produced (glyco-) protein (MAk) to be produced. As these tests are known in the prior art, they are not described here in detail.

[0068] Furthermore, the invention is explained by means of the following examples in connection with the drawings, in which:

[0069]FIG. 1

[0070] The description of a fluidized reactor

[0071] The reactor 2 is partially filled with carrier spheres 4, for example glass spheres of open-pore sintered glass having a diameter of preferably about 0.4 to 0.7 mm, which have undergone an acid treatment (e.g. 2.5 N HCl and 5% nitric acid ) and which, after neutralizing them are subjected to heat treatment for sterilization. (e.g. 220° C. for 6 hrs). Furthermore, the reactor is provided with an aeration module (6) for blowing a gas mixture (8), first into the reactor for whirling around the carrier spheres, and then released again to the outside. The reactor is furthermore provided with pH electrodes, oxygen electrodes temperature probes and similar. The medium is being recirculated in a circulation (10) with flow speed of for example 450 to 500 ml/min. The circulation can be provided with an inlet for the fresh medium (12), a pump (14) and a sample drawing valve (16). From line (18), medium is withdrawn for the harvest of the protein contained therein.

[0072]FIG. 2

[0073] Graphs of results of a modified Lindmo test for the antibody preparations

[0074]FIGS. 3 and 4

[0075] Scintigrams of monoclonal antibody labeled with TC-99m from prior art preparations;

[0076] FIGS. 5 to 7

[0077] Scintigrams of TC-99m labeled monoclonal antibodies from preparations according to the present invention;

[0078] Following are the results of a typical immunoreactivity test for an MAk, as an example recited for MAk BW 250/183.

EXAMPLE 1

[0079] This example is directed to the comparison of the immunoreactivity of MAk BW 250/183 (Eur. J. Nucl. Med. 14 (1988), 523-528) measured with the modified Lindmo-test (see Example 3 for method) at conventional fermentation and purification and the two new fermentation and purification procedures (production method I and production method II) described herein each prior to starting the purification (cell supernatant) and after ending the purification (purified MAk-end product).

[0080] The MAk-containing samples of the cell supernatants respectively the purified MAk-end products were treated, as exactly described in Example 3, and then undergoes the modified Lindmo-test according to the prescribed protocol. An MAk from the identical isotype (IgG₁) was used as a negative control having the same light chain (κ), a comparable isoelectric point and a non-relevant specificity. The positive control is a charge of a MAk BW 250/183 with an immunoreactivity of 91 to 94% purified with an antigen column in an analytic scale.

[0081] Following are the results of each of the immunoreactivity tests of the cell supernatants and the purified MAk end products.

[0082] 1. Conventional Fermentation Method and Conventional Purification Method (Prior Art)

[0083] a) Non-purified Cell Culture Supernatant (ZKÜ)) According to Conventional Fermentation (KF) TABLE 1 Tested Starting Immuno mass in ng mass in ng reactivity negative control — 50.0 — positive control 3.2 48.0 93.3% 250/183, ZKÜ, KF, sample 1 7.5 40.7 81.6% 250/183, ZKÜ, KF, sample 2 8.2 38.6 78.8 % 250/183, ZKÜ, KF, sample 3 8.5 40.2 79.9 % IR medium value of the 79.7 % 3 samples

[0084] The results are shown in the graph of FIG. 2A.

[0085] b) Purified MAk-end Product (MAk) According to Conventional Fermentation (KF) and Conventional Purification (KR) TABLE 2 Tested Starting Immuno mass in ng mass in ng reactivity negative control — 50.0 — positive control 3.2 48.0 93.3% 250/183, Mak, KF + KR, 7.3 24.8 70.6% sample 1 250/183, Mak, KF + KR, 8.1 28.8 71.9% sample 2 250/183, Mak, KF + KR, 8.4 32.4 74.1% sample 3 IR medium value of the 3 72.2% samples

[0086] The results are shown in the graph of FIG. 2B

[0087] 2. Fermentation in the Fluidized Reactor and Conventional Purification Method or “Column Reduced” Purification Method (Invention).

[0088] a) Cell Culture Supernatant (ZKÜ) After Fluidized Fermentation (WF) TABLE 3 Tested Starting Immuno mass in ng mass in ng reactivity negative control — 50.0 — positive control 3.5 45.2 92.4% 250/183, ZKÜ, WF, sample 1 3.7 183.5 98.0 % 250/183, ZKÜ, WF, sample 2 4.0 137.8 97.1 % 250/183, ZKÜ, WF, sample 3 3.9 123.0 96.8 % IR medium value of the 97.3% 3 samples

[0089] The results are shown in the graph of FIG. 2C.

[0090] b) Purified MAk-end Product (MAk) According to Fluidized Bed-fermentation (WF) and Conventional Purification (KR) TABLE 4 Tested Starting Immuno mass in ng mass in ng reactivity negative control — 50.0 — positive control 3.5 45.2 92.4% 250/183, Mak, WF + KR, 3.6 39.5 90.9 % sample 1 250/183, Mak, WF + KR, 4.1 35.6 88.5 % sample 2 250/183, Mak, WF + KR, 3.9 34.6 88.7 % sample 3 IR medium value of the 89.3% 3 samples

[0091] The results are shown in the graph of FIG. 2D.

[0092] c) Purified MAk End-product (MAk) After the Fluidized Bed Fermentation (WF) and “column-reduced” Purification (SR) TABLE 5 Tested Starting Immuno mass in ng mass in ng reactivity negative control — 50.0 — positive control 3.5 45.2 92.4 % 250/183, Mak, WF + SR, 3.5 81.4 95.7% sample 1 250/183, Mak, WF + SR, 3.9 99.5 96.1 % sample 2 250/183, Mak, W + SR, 4.0 100.5 96.0 % sample 3 IR medium value of the 95.9% 3 samples

[0093] The results are shown in the graph of FIG. 2E.

[0094] The end results of Example 1 are summarized in the following Table 6.

[0095] Immunoreactivity of MAk BW 250/183 Production Production conventional Procedure 1 Procedure 2 fermentation Fluidized reactor- Fluidized reactor- procedure and fermentation and fermentation and conventional conventional “column reduced” purification purification Purification method method method Cell culture 80% 97% 97% supernatant Purified MAk 72% 89% 96% final product

[0096] The immunoreactivity of MAk BW 250/183 is distinctly dependent on the fermentation and purification conditions. With conventional fermentation, typically immunoreactivity in the cell culture supernatant up to 80% can be reached. However with the new fermentation method via the fluidized reactor, the immunoreactivity at 97%—is distinctly higher.

[0097] Likewise, the purification method affects the immunoreactivity of MAk. The novel “column-reduced” purification method is distinctly more gentle than the conventional purification method. (8% drop of immunoreactivity with the conventional method as compared to only a 1% drop with the new “column-reduced” purification method).

[0098] Comparable results were realized also with other MAks for example with MAk BW 431/26 (Eur. J. Nucl. Med. 14 (1988), 523-528 and Int. J. Cancer 36 (1985), 75-84), MAk BW 575/931 (Pediatr. Hematol. Oncol. 6 (1989), 73-83), MAk YTH 24.5 (J. Immunol. 134 ( 1985), 3056-3061 and Leucocyte Typing III: White Cell Differentiation Antigens (publ. McMichael et al.), Oxford University Press, Oxford, pp 788-803), MAk BW 278/105 (J. Histochem. Cytochem. 34 (1986), 209-214), the MAk as described in the German patent application 197 44 531.4 as well as the fusion proteins described in British J. Cancer 645, 234-238, 1992. (see table 7). TABLE 7 Immuno Reactivity In Dependence of Fermentation and Purification Method Production Production conventional Method 1 Method 2 fermentation Fluidized reactor- Fluidized reactor- procedure and fermentation and fermentation and conventional conventional “column reduced” Name of MAk purification procedure purification method Purification method BW 431/26, ZKÜ* 85% 96% 96% BW 431/26, GME* 79% 87% 94% BW 575/931, ZKÜ* 75% 93% 93% BW 575/931, GME* 67% 85% 91% YTH 24.5, ZK{umlaut over (Uo)} 83% 98% 98% YTH 24.5,GMEö 77% 89% 96% BW 278/105, ZKÜ* 87% 94% 94% BW 278/105, GME* 81% 85% 92% Fusion Protein ZKU* 84% 97% 97% Fusion Protein GME* 76% 88% 96%

[0099] Since comparable findings were realized with all tested MAks and a very complex (glyco-) protein, the fusion protein (molecular weight of the tetramer under native conditions 500 kDA), it can be assumed that comparable values will be realized with all MAks and (glyco-) proteins that can be fermented in procaryotic and eucaryotic systems and that on that basis, a generalization of the positive results justified.

[0100] The MAk charges produced by conventional methods and purified according to method I and II showed an immunoreactivity after radio-labeling according to the method as described by Schwarz and Steinstrasser (J. Nucl. Med. 28 (1987), 721) an immunoreactivity, which was identical to that of the purified MAk protein in within the scope of the precision as in the Lindmo-test. The data are summarized in the following Table 8: conventional Production Production fermentation Method 1 Method 2 procedure and Fluidized reactor- Fluidized reactor- purified conventional fermentation and fermentation and MaK-final purification conventional “column reduced” product method purification method purification method unmarked 72% 89% 96% Tc-99m 70% 89% 95% marked

[0101] Unexpected Clinical Results

[0102] Aliquots of preparations of the MAk BW 250/183, which were produced either by following conventional production methods (i. e. batch fermentation in stirred fermenters followed by conventional protein chemical purification; immunoreactivity 72%) or, the novel production method II (fluidized reactor followed by “column-reduced” purification method; immunoreactivity 96%) according to GMP were labeled with Tc-99m according to the method of Schwarz and Steinstraesser (J. Nucl. Med., 28 (1987), 721). The portion of the isotope bound to the MAk was in both preparations 99.9%. Patients that were believed to have inflammatory diseases respectively bone marrow metastasized tumors were given a dose of 10-20 m Ci i.v. (intravenously) of the radio-chemical identical MAk-preparation. Full body Scintigrams from dorsal view and frontal view were taken in the time intervals of from 2 to 25 hours after i.v. application of the radio-labeled MAk.

[0103] Surprisingly, it was found from the Scintigrams that after injection with MAk charges that had an immunoreactivity of <90% (production method II) that preferentially the bone marrow was sharply contoured and the spleen was more or less distinctly shown (see Example 3: images GRAN 91, GRAN 81 and GRAN 71). With MAk charges which are produced according to a conventional fermentation and purification method (immunoreactivity 70 to 80%) apart from the bone marrow image, the liver and the spleen were distinctly seen (see Example 3: images GRAN 11 and GRAN 21). Furthermore, theses images show a higher background, which is seen in the Scintigram as slightly foggy and out of focus.

[0104] As a result, the epitope-negative normal tissue of patients, who received MAk charges with an immunoreactivity >90% were burdened substantially less than those patients, who were treated with MAk charges with an immunoreactivity <80%. These observations on patients demonstrate the significance of the level in immunoreactivity of a specific MAk against granulocytes for the images (scintigraphy) in nuclear medicine. The MAk charges with an immunoreactivity >90% show distinct advantages not only in the imaging but also for the therapy with α- and β-emittors. Thus, it is for example possible that after labeling of the MAk charges with an immunoreactivity of ≧90% to couple Re^(188/186), Y⁹⁰ or astatine by methods known from the literature to thus carry out a preferential bone marrow irradiation.

[0105] Within the frame work of a treatment assay, a collective of 19 patients with acute myeloic leukemia or chronic myeloic leukemia were treated. For that purpose charges of the MAk BW 250/183 (immunoreactivity >90%) labeled with Re¹⁸⁸ (specific activity; 5-7.5 GBq/mg) and 6.5-12.4 GBq were applied (i.v.) intravenously.

[0106] Dosiometric research resulted in the data as collected in the following table. TABLE 9 Dosiometric Research after Radio Immunotherapy with Re-188-Iabeld MAk BW 250/183 Date of KM- Organ Dosis in Gy Sex Age Disease Marker Transplantation KM Mz Lb Nie Lu* f 39 AML T (8; 21)  2/98 12.0 7.3 3.8 5.3 n.d. f 38 AML no  3/98 13.0 13.2 6.2 11.3 n.d. m 52 AML no  3/98 8.0 7.0 5.0 11.0 n.d. m 45 AML no  4/98 13.0 12.0 4.0 11.0 n.d. m 50 CML T (12;13)  4/98 12.8 18.6 n.d. 7.6 n.d. f 19 c-ALL no  5/98 5.9 12.3 1.8 7.1 n.d. m 44 c-ALL Ph +  6/98 15.2 12.7 3.2 5.3 0.3 f 17 AML no  7/98 18.4 10.3 4.1 5.0 n.d. m 50 AML T (15; 17)  8/98 10.9 8.6 2.7 4.4 0.4 f 32 AML Deletion  9/98 15.7 6.8 3.0 5.1 0.9 Chrom. 6 m 56 B-CLL no  9/98 15.7 4.0 2.3 4.5 n.d. 1 36 AML no  9/98 13.0 5.6 2.3 15.1 0.6 m 45 c-ALL Ph+ 11/98 6.5 11.5 2.7 4.5 n.d. f 19 AML T (9;11) 11/98 11.4 n.d. 7.2 10.1 1.1 1 40 2. AML no 12/98 13.8 18.2 5.3 10.1 0.3 (MDS) m 51 AML Trisomie 12/98 14.3 6.7 5.9 8.9 0.4 6, 8, 21 Deletion 11q23 m 20 CML Ph+ 12/98 11.9 14.3 7.0 5.5 n.d. 1 47 2. AML T (16; 19) 12/98 16.2 7.6 3.7 5.6 0.5 Plasmo- P (11; 12) cytoma, MDS m* 59 AML no 12/98 19.0 14.0 6.0 8.2 0.7

[0107] The data show, that it is possible to localize high radiation doses on the bone marrow and the spleen of a patient with leukemia by means of radio immunotherapy with the Re-188 labeled MAk BW 250/183 (immunoreactivity >90%)

[0108] The patient group consisted of persons with a relapse risk of 40-50% within 2 years. Surprisingly, the radio immunotherapy induced no significant side effects. After that, a standard therapy followed consisting of 12 Gray full body irradiation sessions and dispensation of cyclophospho amide. All 19 patients could be brought into complete remission, pointing to a future role of radio immunotherapy with MAk BW 250/183 for treating leukemia in conjunction with the standard therapies. It also appears possible that radio immunotherapy will replace the full body irradiation with its negative side effects.

[0109] A further advantage in using MAk B W 250/183 with an immunoreactivity of >90% percent is that the administered amount of 1 mg per application can be reduced to 0.5 mg per application. This reduction leads to an HAMA (human-anti-mouse-antibody)-frequency which was under 2% and which therefore did not deviate significantly from the background HAMA values of normal persons.

[0110] Furthermore, it is surprising that early metastases in RES (reticulo-endothelial systems) of the bone marrow could be discovered with MAk preparations, which had an immunoreactivity of ≧90% that could not definitely be detected when treating with preparations with about 80% immunoreactivity because of over irradiation by lung and liver activity concentration.

[0111] In summary it can be said that the surprising advantages relative to the image quality, diagnostic efficiency, dosiometry and therapeutic efficiency which are generated due the use of high immunoreactive charges of MAk BW 250/183 in connection with α-, β- or γ-emitters were not to be expected either qualitatively or quantitatively but will open the road to a more efficient diagnostic tool of inflammatory processes and of metastases as well as therapies for leukemia and other diseases of the hematopoietic system.

EXAMPLE 2

[0112] Production Method I

[0113] Culturing Eucaryotic Cells and their Fermentation in a Fluidized Reactor

[0114] An ampoule from the working cell bank, stored in liquid nitrogen is being defrosted and the cells contained therein are cultivated under standard cell culture conditions (synthetic protein-free cell culture medium) in T-flasks. When a cell titre of 6-10×10⁷ has been reached, the content of 4-6 T-flasks is further cultivated in a 500 ml-Spinner container under standard cell culture conditions. After reaching cell titre of 1.5-2×10⁸, the cells are then transferred into 2 Spinner containers each of 1000 ml volume and cultivated further. After reaching a cell titre of 1.2-1.5×10⁹, the cells are inoculated in a 900 ml cell culture medium and 200 ml Siran^(R-)carrier containing fluidized reactor (Bioreactor Pilot B 500, Papaspyrou Biotechnologie GmbH, Technologiezentrum Jüilich, D-52428 Jülich) and cultivated in accordance with the directions of the Papaspyrou Biotechnologie company for 60 days at 36.5° C. Oxygen content, pH value, glucose concentration and the MAk-content are being controlled in intervals of 1-4 days. During fermentation the cell culture supernatant is continuously harvested and stored at 4° C.

[0115] Proteinchemical Purification with the Conventional Method

[0116] The volume of the cultured ultraconcentrate to be processed is determined and then 1.5 times the amount of saturated ammonium sulfate slowly added. The suspension remains at 4° C. for up to three days until a clear supernatant is seen. This supernatant is being decanted and the precipitate suspended in the remaining amount of the supernatant and spun off at >5000 g for 30 minutes at room temperature. Sediments are combined in a centrifuge beaker (=>MAk ammonium sulfate paste) and subsequently dissolved with a starting buffer (1 to 4 parts buffer to one part sediment).

[0117] The amount of dissolved sediment which is computed for a protein A run is combined under stirring with a watery Triton X-100 solution, 100 g/l so that the end concentration of Triton X-100 is 0.5 percent (50 ml Triton X-100 solution per 1000 ml sediment in solution). The formulation is subjected to sterile filtration through a spray filter, 0.2 μm. The formulation then remains at rest for 2-18 hours at 4° C. (=>virus inactive MAk solution).

[0118] Immediately thereafter, the protein A fractionation is carried out. The application of the sample is carried out under possibly aseptic conditions and by means of a pump, wherein the flow speed can correspond to a 1-2 fold volume per hour, so that there is contact between MAk and Protein A for at least 30 minutes. After the solutions have been completely applied, the proteins not binding to Protein A and Triton were being washed from the column with starting buffer and are rinsed until the extinction/transmission of the flow-off indicates the beginning value of the starting buffer again (duration: 0.5 to 3 hours). The elution of the MAk is carried out with elution buffer of pH 3.0. The eluate collected in a container cooled with ice flakes (weighed glass bottle) and containing about 0.1 of the column volume of 2 M Tris/HCl, pH 8.0. The elution is carried until the writer indicates that the starting value has been approximately reached again. (=>purified MAk solution).

[0119] After determining the volume, a 1.5 fold amount of saturated ammonium sulfate-solution is added slowly under stirring for 60 minutes. Thereafter the suspension remains standing for up to 3 days at 4° C. until a clear supernatant has formed. The batch is shaken and spun at >5000 g for 30 minutes at room temperature. The supernatant is then decanted and the sediments are combined in a centrifuge beaker. (=>MAk-ammoniumsulfate paste) and dissolved most concentrated in tris/HCl-NaCl-buffer (1-3 parts of buffer to 1 part of sediment).

[0120] The cleared protein solution is immediately applied to a regenerated Sephadex G-25 column equilibrated with Tris/HCl-NaCl-buffer, pH 7.5. The protein solution volume that is to be salt-converted should at the most be 15% of the column volume. The solution coming from the column is fed through a flow-through photometer and subsequently led through a pH-/Ionmonitor. After the MAk solution is completely applied, the column is rinsed with Tris/HCl-NaCl-buffer, pH 7.5 at a flow speed in the column of about 20 ml per cm² per hour. The MAk which runs in the exclusion volume of the column is collected in a sterile receptacle until the line UV writer again indicates the starting value of the extinction/transmission (about 95%). The pH-/Ion monitor is not yet supposed to show a change in conductivity (=>rebuffered MAk-solution).

[0121] The conductivity of the solution is checked and if necessary adjusted to the conductivity of the buffer for injection purposes by adding sodium chloride solution, 10 g/l of water.

[0122] The product is then applied to a prepared Q-Sepharose-column possibly under aseptic conditions and with an application speed of 150 ml per hour (per ml Q-Sepharose 2 to 5 mg protein). The elution is carried out with Tris/HCl-NaCl- buffer, pH 7.5 with a flow speed of about 25 ml per cm² and per hour. The solution coming from the column is fed over a flow-through photometer and subsequently led over a pH-Ion monitor. The MAk flowing from the column is collected in a sterile receptacle according to the UV line writer profile until the line writer indicates approximately the extinction/transmission starting value (about, 95%) (=>de-pyrogenated MAk-solution).

[0123] The pH value of the MAk-containing eluate is adjusted after the anion exchange chromatography with 1N HCl or 1N sodiumhydroxide solution to a pH of 6.9.

[0124] After determining the volume, 1.5 times the amount of saturated ammoniumsulfate-solution is again added slowly under agitation and stirred for 60 minutes at 4° C. Thereafter, the suspension remains standing overnight until a clear supernatant has formed. This formulation is shaken and spun at 8525 g in a centrifuge at room temperature for 60 minutes. Then the supernatant is decanted and the sediments combined in a centrifuge beaker (=>Mak-ammoniumsulfate paste) and dissolved in most concentrated form in sodiumphosphate-NaCl-sorbit-buffer, pH 7.2 (1 to 3 parts buffer to 1 part sediment).

[0125] The cleared protein solution is immediately applied under possibly aseptic conditions (at most 15% of the column volume) to a regenerated G-25 Sephadex column which has been equilibrated with sodiumphosphate-NaCl-sorbit buffer, pH 7.2. The solution coming from the column is led over a flow-through photometer and subsequently over a pH-/ion monitor. The column is then rinsed with sodium phosphate-NaCl-sorbit buffer, pH 7.2 (flow speed about 20 ml per cm² and per hour). The MAk which comes from the exclusion volume of the column is collected in a sterile container according to the UV-line writer profile until the line writer indicates again approximately the extinction/transmission starting value (about 95%). The pH-/ion monitor should not yet indicate a change in the conductivity (=>MAk-bulk solution, concentrated)

[0126] The pH value of the solution is checked and adjusted to pH 7.2 with 1N HCl or 1 N sodiumhydroxide solution.

[0127] By means of the values for the mouse IgG-concentration and the volume, the “final bulk” is diluted with sodiumphosphate-NaCl sorbit-buffer, pH 7.2 to the desired final concentration.

[0128] The product is then sterile-filtrated over a 0.2 μm-one-time filter and divided into aliquots and stored as the “final bulk” at −20° C.

[0129] Production Method II

[0130] Culturing of Eucaryotic Cells and their Fermentation in a Fluidized Reactor

[0131] An ampoule from a working cell bank stored in liquid nitrogen is defrosted and the cells contained therein cultivated according to standard cell culture conditions (synthetic protein-free cell culture medium) in T-flasks. After the entire cell count has reached 6-10×10⁷, the content of 4-6 T-flasks is further cultivated in a 500 ml Spinner container under standard cell culture conditions. After the cell count reaches 1.5-2×10⁸, the cells are transferred to 2 Spinner containers each with 1000 ml volume and further cultivated. After reaching a cell count of 1.2-1.5×10⁹, the cells are inoculated in a fluidized reactor (Bioreactor Pilot B 500, Papaspyrou Biotechnologie GmbH, Technologiezentrum Jülich, D-52428 Jülich) containing 900 ml cell culture medium and 250 ml Siran^(R) carrier and cultivated in accordance with the methodology of the Papaspyrou Biotechnologie GmbH for 60 days at 36.5° C. Oxygen content, pH value, glucose concentration and MAk-content are checked in intervals of 1 to 4 days. During the fermentation, the cell culture supernatant is continually harvested and stored at4° C.

[0132] Protein-Chemical Purification with a “Column-reduced” Method.

[0133] After harvesting 18 l cell culture medium, a cell separation follows by means of “tangential flow” micro filtration followed by a sterile filtration through a 0.2 μm filter. The cell-free and sterile cell culture harvest is stored at 20° C. until sufficient cell culture harvest was collected for a subsequent purification (cell culture harvest of about 250 l cell culture medium). After the desired batch size has been reached, the sterile cell culture harvest is defrosted, cleared through microfiltration and concentrated 100 to 150 times by means of ultra filtration. Thereafter, the ultra concentrate is combined with the same volume of 2 times concentrated starting buffer (pH8.6) and sterile filtrated. A sterile Triton X-100 solution (100 g Triton/l) ad 0.5% Triton end concentration is added under stirring. The solution is left standing for 4 to 18 hours at 4° C. in order to inactivate the coat-containing viruses.

[0134] Thereafter, the ultraconcentrate treated with Triton is pumped onto a Protein A-Sepharose-4-fastflow column, which was equilibrated previously with 5 times the column volume of starting buffer. Proteins that are non-binding and Triton X100 are washed from the column with 5 column volumes starting buffer. Subsequently, MAk linked to the A-protein is washed from the column with the elution buffer (pH 3.0) and collected in 130 ml neutralization buffer (pH 8).Thereafter, the MAk which has been collected in neutralization buffer is adjusted to a pH of 7-7.5 with NaOH depending on need, then diluted with the same volume of 2× membrane adsorbing buffer (pH 7.5) and sterile filtrated. Subsequently, the MAk solution is pumped under sterile conditions through a membrane adsorber, which was previously equilibrated with WFI and the adsorption buffer (pH7.5). The flow-through containing the MAk now free from contaminating pyrogens and DNA is collected in a sterile container and adjusted with buffer (pH 7.5) to 500 μg MAk/ml. Thereafter the diluted MAk solution undergoes ultrafiltration with a Viragard-hollow fiber module to remove potentially contaminating viruses. The permeate which contains the virus-free MAk is concentrated by means of ultrafiltration to a concentration of 4-5 mg MAk/ml and diafiltrated against an endbuffer (pH 7.2). After sterile-filtration, dilution by end buffer and renewed sterile-filtration the MAk is stored as bulk material.

EXAMPLE 3 Modified Quantitative Immunoreactivity Test According to Lindmo

[0135] Introduction:

[0136] In order to determine the content of immunnoreactive monoclonal antibodies in hybridoma supernatants, a binding assay with an excess amount of antigen in combination with a sensitive (1-2 ng mouse-Ig/ml) ELISA-system was used for the determination of the portion of unbound monoclonal antibodies. This test has essentially two advantages as compared with the immunoreactivity test developed by Lindmo, namely

[0137] a) the test permits determining the immunoreactivity of un-purified as well as purified non-radio labeled and radio labeled MAk; and

[0138] b) the test permits determining the immunoreactivity in the absence of unspecific binding.

[0139] Material

[0140] 1.1 Chemicals and Materials Name Manufacturer Order No. Formaldehyde solution, 37% Merck 818708 Sodium dihydrogen phosphate-1-hydrate Merck 6346 Di-sodiumhydrogen -phosphate-2-hydrate Merck 30412 PBS Behringwerke Glycin Merck 104201 96 well micro titre plates, Type B Nunc 4-60445 Goat-anti-mouse-IgG (“catcher”) Sigma M8642 Tween PBS for Enzygnost Behringwerke OSWE96 Casein Sigma C5890 Goat-anti-mouse IgG,-antibody coupled with alkaline phosphates SBS 107-04 4-methyl-umbelliferyl-phosphat Sigma M 8276 SDS Sigma L 5750

[0141] 1.2 Instruments Centrifuge Heraeus Minitubes, 1.0 ml Kühn & Bayer 64698446 Rotation instrument Heidoiph Reo x2 Analytical scale Mettler DE 100 Magnetic stirrer IKA RCT pH-meter WTW Moulinette moulinex Fluoroskan Merlin

[0142] Preparation of Solutions Needed

[0143] 4% Formaldehyde Solution According to Lilly

[0144] to 100 ml 37% formaldehyde solution,

[0145] 900 ml aqua bi-distilled is added. In this solution

[0146] 4 g sodium hydrogen diphosphate and

[0147] 6.5 g di-sodium hydrogenphosphate are dissolved under stirring.

[0148] The pH value of this solution is pH 7.0

[0149] Rinsing Solution: 0.05 Tris-Citrate-Buffer pH 7.4

[0150] 6.06 gTris

[0151] 19,5 g citric acid-1-hydrate and

[0152] 4.25 sodium hydroxide

[0153] are dissolved ad 1 l aqua bi-distill.

[0154] Blocking Solution: 1% Casein in PBS, pH 7.2

[0155] 10 g casein in

[0156] 1 l cold PBS, pH 7.2+phenol red

[0157] stir 30 minutes

[0158] thereafter spin at 3000 rpm and

[0159] filter supernatant through a folding filter.

[0160] pH-value of the solution is adjusted with NAOH

[0161] Substrate Buffer: 0.5 Tris, 0.01% MgCl, pH 9.6

[0162] 60,57 g Tris and

[0163] 0.1 g magnesiumchloride are dissolved

[0164] add 1 l aqua bi-distill.

[0165] 4-Methyl-Umbelliferyl-Phosphate (MUP) Solution

[0166] concentration of the MUP solution is 1 mg 4-MUP/4 ml

[0167] substrate buffer.

[0168] Stop-Solution: 0.2 M Glycine. 02.% SDS. pH 11.7

[0169] 15 g glycine and

[0170] 2 g SDS are dissolved in

[0171] ad 1 l aqua bi-distill.

[0172] The pH value of the solution is adjusted with 5N NaOH.

[0173] Modified Quantitative Immunoreactivity Test According to Lindmo

[0174] 1. Production of the Antigen Containing Materials (“Antigen Containing Material”, ACM)

[0175] Tissue of human Tumor-Xenografts (MZ-STO 1, stomach carcinoma), which express the epitope of the BW 250/183 (at the membrane of granulocytes expressed “non-specific cross reacting antigen” (NCA-95)) are being cut up in a Moulinette into 2 to 5 mm pieces and

[0176] fixed according to Lilly in 4% formaldehyde solution for at least 16 hours at room temperature.

[0177] After rinsing, the fixed tissues are passed through a stainless steel strainer.

[0178] The fixed cells are washed at least 10 times with PBS until the supernatant is approximately clear and then

[0179] washed once with formalin solution.

[0180] This preparation is stored at 4° C. in formalin solution according to Lilly (1 Part ACM and 1 part formalin according to Lilly) and designated as “ACM”.

[0181] 2. Binding-Assay with Antigen Excess

[0182] The ACM is washed at least 10 times with PBS and subsequently,

[0183] the pellet is suspended and incubated in a 100 mM glycine (4-times the pellet volume) for 30 minutes at 4° C.

[0184] Thereafter, the cells are again washed 4 times with PBS.

[0185] Increasing amounts of ACM are (0.1 up to 50 mg) are put in 1 ml minitubes and each minitube incubated with 500 μl hybridoma supernatant containing 25 ng MAk BW 250/183 overnight at room temperature (overhead-rotation).

[0186] Negative controls are incubated with 25 ng of the anti-mycoplasm MAk BW 227/7 having the same isotype (IgG₁).

[0187] ACM is spun off;

[0188] Supernatant is removed and

[0189] the remaining mouse-IgG-molecules which are not bound to the ACM pellet are then analyzed.

[0190] 3. Determination of the Portion of Unbound Mouse-IgG-Molecules in the ELISA-System

[0191] Coating the Micro-titre Plates with Antigen

[0192] 96-well polystyrol-micro-titre plates are incubated at room temperature with 50 μl goat-anti-mouse-IgG-antiserum, 2.5 μl/ml, per well over night.

[0193] The rabbit-anti-mouse-IgG-antiserum is subsequently aspirated and the plates are washed 4 times with 0.5 M Tris-citrate buffer, pH 7.4 (1 washing round=200 μl washing solution per well pipetting and aspirating)

[0194] The micro-titre plates are dried overnight, inverted on cellulose.

[0195] The shelf life of the so pretreated micro-titreplates which are sealed in dry cartridges is at least 6 months.

[0196] Blocking of Free Binding Sites

[0197] 200 μl blocking solution are pipetted per well and the plates are incubated at room temperature for 60 minutes.

[0198] Subsequently, the blocking solution is aspirated

[0199] Application of the Sample

[0200] 50 μl of the ACM supernatants, to be analyzed for the number of unbound mouse-IgG-molecules, are applied per well and incubated at room temperature for 60 minutes.

[0201] Thereafter, the micro-titre-plates are washed 3 times as afore-described with washing solution.

[0202] Amplification and Detection

[0203] 50 μl of a goat-anti-mouse-IgG-antibody which has been coupled with alkaline phosphatase diluted at a ratio 1:250, is applied per well and incubated at room temperature for 30 minutes.

[0204] The micro-titre plates are washed 3 times with washing solution as afore-described.

[0205] 50 μl MUP solution is applied per well and incubated at room temperature for 30 minutes.

[0206] The substrate reaction is stopped after the incubation at room temperature for 30 minutes by adding 100 μl stop solution.

[0207] Thereafter, fluorescence is measured: the start-up wavelength is 355 nm and the emission wavelength is 460 nm.

[0208] The test has a sensitivity in the range of 1 to 2 ng mouse IgG/ml.

[0209] Mathematical Determination of the Immunoreactivity

[0210] IR [%]=100%-[100%×(MAk-concentration in supernatant/MAk-starting concentration.)]

[0211] The computation of the immunoreactivity is carried out at the reading point. This point in the curve corresponds to that volume of ACM (X axis of the ELISA-curve) where a drop in the plateau value for the non-specific control (non-specific binding) has not yet shown up.

EXAMPLE 4

[0212] Scintigrams Taken After Injection of Tc-99m Labeled Mak BW 250/185

[0213] The MAk charges utilized for the labeling with Tc-99m, vary in their immunoreactivity due to the type of production method. Data for these production methods are summarized in the following table 10 Production MAk Charge Method Immunoreactivity Scintigram MAk Conventional 70-80 % Image GRAN 11 BW 250/183 fermentation (FIG. 3) method and Image GRAN 21 conventional (FIG. 4) purification method MAk Fluidized reactor >90 % Image GRAN 91 BW 250/183 fermentation and (FIG. 5) “column reduced” Image GRAN 81 method (FIG. 6) Image GRAN 71 (FIG. 7) 

1. Preparation of immunoreactive proteins, characterized in that the percentage of immunoreactive molecules as determined in a modified Lindmo-test is >81% relative to the total number of molecules.
 2. Preparation according to claim 1, characterized in that the percentage of immunoreactive molecules as determined in the modified Lindmo test is >90%.
 3. Preparation according to claim 1 or 2, characterized in that the immunoreactive proteins are produced by cell culturing, in particular by expression in recombinant eucaryotic host cells.
 4. Preparation according to anyone of claims 1 to 3, characterized in that the immunoreactive proteins are labeled, in particular with a radioactive marker.
 5. Preparation according to anyone of claims 1 to 4, characterized in that the immunoreactive proteins comprise at least one antibody-binding domain.
 6. Preparation according to anyone of claims 1 to 4, characterized in that the immunoreactive proteins are selected from antibodies and antibody fragments.
 7. Preparation according to anyone of claims 1 to 4, characterized in that the immunoreactive proteins are selected from fusion proteins, comprising at least one antibody binding domain and at least one effector domain.
 8. Preparation according to anyone of claims 1 to 7, characterized in that the immunoreactive protein binds to an epitope on the cell membrane of malignant and/or non-malignant cells.
 9. Preparation according to anyone of claims 1 to 5, characterized in that the immunoreactive protein binds to a cytoplasmic or extracellular epitope on normal and/or malignant cells.
 10. Preparation according to anyone of claims 1 to 4, characterized in that the immunoreactive protein is a monoclonal antibody which binds to an epitope at CD66, preferably CD66 a, b, c, e and especially preferred at CD66 e.
 11. Preparation according to anyone of claims 1 to 4, characterized in that the immunoreactive protein is a monoclonal antibody that binds to an epitope at CD45.
 12. Preparation according to anyone of claims 1 to 4, characterized in that the immunoreactive protein is a monoclonal antibody that binds to an epitope at N-CAM.
 13. Preparation according to anyone of claims 1 to 4, characterized in that the immunoreactive protein is a monoclonal antibody that binds to an epitope at the VEGF/VEGF-receptor-complex, and which does neither bind to VEGF nor to the VEGF receptor.
 14. Preparation according to anyone of claims 1 to 4, characterized in that the immunoreactive protein is a monoclonal antibody selected from BW 431/26, BW250/183, YTH 24.5 and BW278/105.
 15. Method for preparing a preparation of immunoreactive proteins characterized in that it comprises a fluidized reactor fermentation of a host cell which expresses the immunoreactive protein in a suitable culture medium, and the recovering of the protein from the host cell and/or from the culture medium.
 16. Method according to claim 15 characterized in that the host cell is a eucaryotic cell.
 17. Method according to claim 15 or 16 characterized in that the immunoreactive protein is obtained from the host cell and/or the culture medium by means of a “column-reduced” purification procedure.
 18. Method according to claim 16 characterized in that the immunoreactive protein is obtained from the host cell and/or the culture medium by means of a conventional purification procedure.
 19. Method according to anyone of claims 15 to 18 characterized in that the percentage of functionally active molecules as tested by a modified Lindmo-test is >81% relative to the total number of molecules.
 20. Method according to anyone of claims 15 to 19 characterized in that the percentage of functionally active molecules as tested by the modified Lindmo-test is >90% relative to the total number of molecules.
 21. Method according to claim 18 characterized in that the preparation is produced according the production method I.
 22. Method according to claim 17 characterized in that the preparation is produced according the production method II.
 23. Method according to anyone of claims 15 to 22 wherein the protein is a monoclonal antibody selected from BW 250/183, BW 413/26, YTH 24.5 and BW 278/105 or a comparable murine, humanized, recombinant manipulated monoclonal antibody or fragments thereof.
 24. Method according to anyone of claims 15 to 22 wherein the protein is a monoclonal antibody which binds to an epitope at the VEGF/VEGF receptor complex and which does neither bind to VEGF nor to the VEGF receptor, or a comparable murine, humanized, recombinant manipulated monoclonal antibody or a fragment thereof.
 25. Method according to anyone of claims 15 to 22 characterized in that the protein is a fusion protein comprising at least one antibody binding domain and at least one effector domain.
 26. Use of protein preparations according to anyone of claims 1 to 14, as a carrier of γ-radiators, such as for example Tc-99m, for the production of an agent for diagnosis of inflammatory processes and bone marrow displacing processes, for example metastases of prostrate carcinoma, mamma carcinoma and lymphoma.
 27. Use of the protein preparation according to anyone of claims 1 to 14, as a carrier of α- and β-radiators for the production of an agent for therapy of diseases of the hematopoietic system, preferably leukemia. 